Our specific aims are to characterize the cellular systems operating to repair DNA damage induced in the eukaryote Saccharomyces cerevisiae following treatment of cells with 4,5',8-trimethylpsoralen plus irradiation with 360 nm light. In order to achieve these aims, we will carry out the following experiments. We will quantitate crosslinks in the nuclear and mitochondrial DNA by a sensitive and rapid technique using S1 nuclease. Treatment of cells with khellin followed by irradiation with 360 nm light will be used to assess the contribution of monoadducts to survival, repair and genetic recombination, since khellin, unlike 4,5',8-trimethylpsoralen, produces mainly monoadducts in the DNA. We will determine the rate of excision of crosslinks from both nuclear and mitochondrial DNA in wild type and various repair defective mutants. Excision of crosslinks from nuclear DNA may involve different gene functions than excision of UV induced pyrimidine dimers. Single strand breaks in nuclear DNA resulting from excision of crosslinks and their rejoining will be studied in wild type and rapair defective mutants by sedimentation of DNA from treated cells in alkaline sucrose gradients. In order to determine if the rejoining of strand breaks in nuclear DNA involves recombination between strands of sister duplexes, density and radioactive isotope labeling experiments will be carried out. Differential ability to repair crosslinks from nuclear DNA in synchronized haploid cells in G1, S and G2 phases will indicate whether a duplicated genome is required for effective repair in haploids. Crosslink repair will also be studied in a/a, a/a and a/a wild type and various repair defective diploids since different repair mechanisms may operate in a/a vs. a/a or a/a diploids. We will also determine the effectiveness of crosslinks as compared with monoadducts in inducing genetic recombination. Both homologous recombination in diploids and sister chromatid recombination in haploids will be examined in wild type and various repair defective mutants after 4,5',8-trimethylpsoralen + light treatment. Since several human genetic disorders involve defective DNA repair, an understanding of how cells respond to DNA damage should provide useful information in treating these diseases.